SWITCHING)ON)THE) POWER)OF) E.#COLI# - - PowerPoint PPT Presentation

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SWITCHING)ON)THE) POWER)OF) E.#COLI# - - PowerPoint PPT Presentation

Feb 13, 2024 44 likes •527 views

SWITCHING)ON)THE) POWER)OF) E.#COLI# A"customisable"integrase"switch" Water"Purifica5on" Produc5on"of"clean"water"using"biological"processes"

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A"customisable"integrase"switch"

SWITCHING)ON)THE) POWER)OF)E.#COLI#

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  • Produc5on"of"clean"water"using"biological"processes"
  • Use"in"agriculture"and"industry"
  • Possible"source"of"drinking"water"

Water"Purifica5on"

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  • Bacteria"have"been"engineered"to"uptake"or"produce"

different"substances"

  • Removing"the"bacteria"from"the"liquid"is"challenging"

Our"Mo5va5on"

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The"Idea:"Inducing"Gas"Vesicles"

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  • IntegraseIbased"inversion"

switch"

  • Inheritable"and"inducible"

switch"mechanism"" "

  • Enable"flota5on"

"

"

How"Can"We"Induce"Them"To"Float?"

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SLIDE 6

What"Did"We"Do?"

  • Public"engagement"
  • Model"
  • Swimming"KO"
  • Plasmid"rescue"of"the"knockouts"
  • Created"Gvp/promoter"constructs"
  • Created"an"RFP/GFP"inversion"switch"ac5vated"by"integrase"

" "

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  • Understanding"the"public’s"view"of"GM"organisms"
  • Helped"to"focus"the"direc5on"of"the"project"
  • Allowed"star5ng"point"from"which"to"contact"professionals"in"the"field"
  • Helped"to"introduce"synthe5c"biology"to"children"in"a"fun"way"

Glasgow"Science"Centre"2014"

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Gate"1"–" Idea" Stage"1"–"Developing" the"Basics" Gate"2"–"Choosing" Applica5on" Stage"2"–"Developing"Final" Technology" Gate"3"–"Suitable"for" Industry?" Stage"3"–" Scaling"Up" Gate"4"–"Considering" the"Future" Industrial"Use"of" Product"

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Outreach"Results:"The"Public’s"View"

Would"you"be"comfortable" "with"using"our"bacteriaItreated"water?""

n"="100"

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What"Did"We"Do?"

  • Outreach"
  • Model"
  • Swimming"KO"
  • Plasmid"rescue"of"the"knockouts"
  • Created"Gvp/promoter"constructs"
  • Created"an"RFP/GFP"inversion"switch"ac5vated"by"

integrase" " "

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Bacteria"and"Buoyancy"

ρ))=)250kg/ m3)

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Run"and"Tumble"

Based"on"a"Random"Walk"

  • Run"Time"="Exponen5al"Distribu5on:"
  • Mean:"1"second"
  • Tumble"angle"="Normal"

Distribu5on:"

  • Mean"="68°"
  • Standard"Devia5on:"36°"

""

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Buoyancy"And"Run"And"Tumble"

Results":"When"bacteria"swim,"buoyancy"is"not"a"significant"force"–" bacteria"do"not"‘float’."" Conclusion:"floa5ng"requires"knocking"out"swimming.""

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What"Did"We"Do?"

  • Outreach"
  • Model"
  • Swimming"KO"
  • Plasmid"rescue"of"the"knockouts"
  • Created"Gvp/promoter"constructs"
  • Created"an"RFP/GFP"inversion"switch"ac5vated"by"

integrase" " "

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  • Poten5al"mo5lity"gene"candidates"

"

Mo5lity"Genes"

Modified"from"Reid"2006"

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MotA"Gene"Rescue"of"KO"Strain"

WildItype"DS941" DS941"MotA"KO" DS941"MotA"KO"+"promoter/MotA"

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MotA"Gene"Rescue"of"KO"Strain"

WildItype"DS941" DS941"MotA"KO" DS941"MotA"KO"+"promoter/MotA"

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motA% motB%

motA% motB%

RBS" ACCGAGGAAGCATGAGAATCAAG M K N Q T E E A *

motA% motB%

eryR"

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MotA/MotB"Gene"Rescue"of"KO"Strain"

WildItype"DS941" DS941"MotA"KO" DS941"MotA"KO"+"promoter/MotA/MotB"

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MotA/MotB"Gene"Rescue"of"KO"Strain"

WildItype"DS941" DS941"MotA"KO" DS941"MotA"KO"+"promoter/MotA/MotB"

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MotA/MotB"Gene"Rescue"of"KO"Strain"

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FliC"Gene"Rescue"of"KO"Strain"

WildItype"DS941"" DS941"FliC"KO" DS941"FliC"KO"+"promoter/FliC"

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FliC"Gene"Rescue"of"KO"Strain"

WildItype"DS941"" DS941"FliC"KO" DS941"FliC"KO"+"promoter/FliC"

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FliC"Gene"Rescue"of"KO"Strain"

0" 1" 2" 3" 4" 5" 6" 7" 8" 9" WT" FliC"KO" FliC"+"no"promoter" J23116"+"FliC" J23106"+"FliC"

Distance"(cm)"

FliC"Mo5lity"Swarm"Assay""

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What"Did"We"Do?"

  • Outreach"
  • Model"
  • Swimming"KO"
  • Measurement"system"
  • Created"gas"vesicle"protein"(Gvp)/promoter"constructs"
  • Created"an"RFP/GFP"inversion"switch"ac5vated"by"

integrase" " "

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Floa5ng"Efficiency"Measurement"System"

  • 1. Power"supply"
  • 2. Breadboard"with"2"LEDs"
  • 3. Light"Blocker/Diffuser"

Unit"

  • 4. Cuvene"holder"unit"
  • 5. Webcam"
  • 6. Mouldable"Adhesive"

(Blutac)" "

Based"on"light"scanering"by"bacteria.""

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Measurement"System:"Results"

  • Used"yellow"

glass"" """""beads"(5μm)" "

  • Close"to"

""""theore5cal" """"speeds"but:" """"Sample)size)was" limited""

[NaCl]) Average)Speed) (μm/s)) TheoreKcal)at) 20°C)(μm/s)) 1M" 12.1" 12.0" 3M" 12.7" 9.0" 5M" 8.2" 6.1"

Height"(cm)" 3" 0"

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What"Did"We"Do?"

  • Outreach"
  • Model"
  • Swimming"KO"
  • Measurement"system"
  • Created"gas"vesicle"protein"(Gvp)/promoter"constructs"
  • Created"an"RFP/GFP"inversion"switch"ac5vated"by"

integrase" " "

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  • GvpA"and"GvpC"from"

Planktothrix%rubescens%

  • GvpA"

! Main"component"of"GV" ! Single"pep5de"wall%

  • GvpC"

! Strengthens"GvpA"wall"

Gas"Vesicle"Genes"

Walsby"1994"

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GVP"Construct"

  • We"added"ribosome"binding"sites"biobricks"

to"exis5ng"gvpA"and"gvpC"biobricks"

  • We"then"made"a"composite"biobrick"with"

gvpA"and"gvpC"together.""

  • We"put"these"under"the"control"of"an"

arabinose"inducible"promoter"

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Arabinose"+" Arabinose"I"

Gas"Vesicle"Protein"Expression"

GVP"expression"appears"toxic"

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Arabinose"+" Arabinose"I"

Gas"Vesicle"Protein"Expression"

GVP"expression"appears"toxic"

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What"Did"We"Do?"

  • Outreach"
  • Model"
  • Swimming"KO"
  • Measurement"system"
  • Created"gas"vesicle"protein"(Gvp)/promoter"constructs"
  • Created"an"RFP/GFP"inversion"switch"ac5vated"by"

integrase" " "

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An"IntegraseIBased"Inversion"Switch"

Viral"protein"for"genome"integra5on" " Binds"to"a5P"and"a5B%and%it"inverts"the"sequence"between"the"sites"

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An"IntegraseIBased"Inversion"Switch"

Viral"protein"for"genome"integra5on" " Binds"to"a5P"and"a5B%and%it"inverts"the"sequence"between"the"sites"

We"have"created"a"customisable"switch"biobrick"that"anyone"can"use"to"control" genes"of"their"choice.""

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Reverse"Ribosome"Binding"Site"

Part)Number) Predicted)Binding)Strength)(au)) BBa_B0034) ~20,000" BBa_I742130) ~500" BBa_K1463560) ~12,000"

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Integrase"

Switch"Characterisa5on"with"fluorescent"proteins"

BBa_K1463000:"Switch"Biobrick"

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Our"Switch"Works"

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Our"Switch"Works"

integrase"

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Our"Switch"Works"

integrase"

arabinose)

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In"Conclusion"

  • Implemented"a"stage"gate"approach"that"focused"our"project"on"

fresh"producing"water"for"agriculture.""

  • Created"a"customisable"integraseIbased"inversion"switch"in"a"

biobrick"

  • Showed"that"our"switch"worked"with"RFP"and"GFP"
  • Created"a"strong"reverse"ribosome"binding"site"
  • Informed"by"models"of"bacterial"swimming"and"floa5ng"
  • Knocked"out"mo5lity"genes""
  • Created,"characterised"and"submined""

"I"biobricks"containing"2"different"mo5lity"genes" "I"a"biobrick"containing"2"gas"vesicle"genes"

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What"Now?"

  • Further"develop"gas"vesicle"produc5on"
  • Assemble"and"characterise"the"final"construct"
  • Other"applica5ons"of"the"switch"
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SLIDE 43
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Jake"Casson,"Gintarė"Sendžikaitė,"Aimee"Bias,"Mar5n"Campbell,"Beth"Greig,"Jacob"Roberts," Lydia"Alldred,"Robbie"Evans,"Amy"Ferguson"and"Gemma"McLelland"

The"2014"Glasgow"iGEM"Team"

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Many"Thanks"To"

  • Dr"Sean"Colloms"
  • Professor"Marshall"Stark"
  • Dr"Julien"Reboud"
  • Dr"Joseph"Gray"
  • Dr"John"Walls"
  • Dr"Femi"Olorunniji"
  • Dr"Annegret"Honsbein"
  • Professor"Anna"Amtmann"
  • Ms"Emma"Smith"
  • Ms"Jumai"Adeola"Abioye"
  • Ms"Jia"Zhao"
  • Dr"Saikat"Jana"
  • Ms"Nadia"Colloms"
  • Mr"Jamie"Gallagher"
  • Ms"Hayley"Macfarlane"
  • Mr"Kevin"Dorman"
  • The"iGEM"Teams"involved"in"SULSA"Conference"and"London"Meet"Up"
  • Bower"Building"Staff"
  • Glasgow"Science"Centre"Staff"and"Patrons"
  • Wolfson"Medical"Building"Staff"
  • The"Ins5tute"of"Molecular,"Cell"and"Systems"Biology"
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Our)Sponsors)

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Any"ques5ons?"

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References"

Eight"of"Fourteen"gvp"Genes"Are"Sufficient"for"Forma5on"of"Gas"Vesicles" in"Halophilic"Archaea,"Offner."S.,"Hofacker."A.,"Wanner."G.,"Pfeifer."F.," 2000,"Journal"of"Bacteriology,"Vol"182,"No"15,"pg"4328I4336" Distribu5on,"forma5on"and"regula5on"of"gas"vesicles,"Pfeifer."F.,"2012," Nature"Reviews,"Microbiology,"Vol"10,"pg"705I715" Quorum"sensingIcontrolled"buoyancy"through"gas"vesicles,"Ramsay."J.P." and"Salmond."G.P.C.,"2011,"Communica5ve"&"Integra5ve"Biology,"Vol"5," Issue"1."pg"96I98" Benenson,"Y."(2013)."Recombina5on"Logic."Science,"340."P"554I555"" Wang,"Y,"Yau"Y"Y,"PerkinsIBalding,"D,"Thomson,"J"G."(2011)."integrase" technology:"applica5ons"and"possibili5es."Plant"Cell"Rep,"30."P"267–285" Dillon,R."Fauci,L."Gaver,D."A"Microscale"Model"of"Bacterial"Swimming," Chemotaxis"and"Substrate"Transport.Journal"of"Theore5cal"Biology.1995." Vol"177(4)."p"325I340.DOI:"10.1006/jtbi.1995.0251."(accessed"July"2014)" Reid,"S.,"Leake,"M.,"Chandler,"J.,"Lo,"C.,"Armitage,"J."and"Berry,"R."(2006)." The"maximum"number"of"torqueIgenera5ng"units"in"the"flagellar"motor"

  • f"Escherichia"coli"is"at"least"11."Proceedings%of%the%Na:onal%Academy%of%

Sciences,"103(21),"pp.8066II8071."